Selection and Mechanism Study of Q-Markers for Xanthocerais lignum Anti-Rheumatoid Arthritis Based on Serum Spectrum–Effect Correlation Analysis

Objective: To elucidate the chemical profile of Xanthocerais lignum’s extracts of different polarities and their impact on rheumatoid arthritis (RA), we identified anti-RA markers and predicted their action mechanisms. Methods: A collagen-induced arthritis rat model was established, and UPLC-Q-Exactive Orbitrap MS technology was employed to analyze and identify the chemical constituents within the alcohol extract of Xanthocerais lignum and its various extraction fractions, as well as their translocation into the bloodstream. Serum spectrum–effect correlation analysis was utilized to elucidate the pharmacodynamic material basis of Xanthocerais lignum against RA and to screen for Q-Markers. Finally, the potential anti-RA mechanisms of the Q-Markers were predicted through compound-target interaction data and validated using molecular docking techniques. Results: We identified 71 compounds, with flavan-3-ols and flavanones as key components. Of these, 36 were detected in the bloodstream, including 17 original and 19 metabolized forms. Proanthocyanidin A2, dihydroquercetin, catechin, and epicatechin (plus glucuronides) showed potential anti-RA activity. These compounds, acting as Q-Markers, may modulate ERK, NF-κB, HIF-1α, and VEGF in the HIF-1 pathway. Conclusions: This research clarifies Xanthocerais lignum’s pharmacodynamic material basis against RA, identifies 4 Q-Markers, and offers insights into their mechanisms, aiding quality assessment and lead compound development for RA treatment.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disorder primarily characterized by erosive, symmetrical polyarthritis, frequently afflicting smaller joints such as those in the hands and feet, and potentially extending to extra-articular systems, even culminating in joint deformities and functional impairment [1].Untreated, RA significantly diminishes patients' quality of life and life expectancy.The global incidence of RA is approximately 0.5% to 1.0%, with a peak onset age between 30 and 50 years, and the prevalence in females is roughly 3 times that of males [2].
Currently, the scientific community is actively engaged in the quest for alternative natural remedies with anti-RA properties derived from plants, animals, and microorganisms.These medicinal sources exhibit a wide range of diversity and abundance, coupled with minimal adverse effects.Studies on their mechanisms of action have revealed that many of their constituents possess anti-inflammatory and immunomodulatory properties, offering distinctive advantages in the treatment of RA [3,4].Xanthocerais lignum is the dried stem or branch wood of Xanthoceras sorbifolia Bunge in the Sapindaceae family, as recorded in classical Mongolian and Tibetan medical texts such as "Ren Yao Bai Jing Jian", "Wu Wu Meng Yao Jian", "Jing Zhu Ben Cao", and "Meng Yao Zhi".It is known for its efficacy in "Zao Xie Ri Wu Su", clearing heat, reducing swelling, and alleviating pain, primarily used clinically to treat RA and rheumatic internal heat [5][6][7].
Quality markers (Q-Markers) in traditional Chinese medicine are indicative substances reflecting the safety and efficacy of Chinese herbs, based on the core theory of "Chinese medicine efficacy-pharmacodynamic material basis-quality control characteristic components" [8].Chinese medicine, a complex multi-component system, often presents challenges in identifying and analyzing its pharmacodynamic material basis using traditional methods.Serum spectrum-effect correlation analysis is an emerging research approach that considers the metabolism and transformation of Chinese medicine within the body, identifying the translocated components of Chinese medicine in the blood and correlating them with pharmacological data to pinpoint chromatographic peaks associated with efficacy [9].This method not only reveals the synergistic actions of multiple targets and components inherent in Chinese medicine but also embodies the holistic perspective of traditional Chinese medicine.The anti-RA therapeutic efficacy of Xanthocerais lignum is well-established, yet its pharmacodynamic material basis and mechanisms of action remain unclarified.Therefore, this study aims to elucidate the chemical constituents and constituents absorbed into the blood of Xanthocerais lignum using UPLC-Q-Exactive Orbitrap MS technology and to perform serum spectrum-effect correlation analysis with its anti-RA efficacy in order to screen for anti-RA Q-Markers of Xanthocerais lignum.Furthermore, this study predicts the mechanisms of action of these Q-Markers based on compound-target interaction data and validates them through molecular docking techniques.

Chemical Constituents of Various Extracts of Xanthocerais lignum
The total ion current (TIC) graphs of the various extracts of Xanthocerais lignum in negative and positive ion modes are shown in Figures 1 and 2, respectively.A comparison reveals that there are more chromatographic peaks and better chromatographic separation and signal strength in the negative ion mode.The ethanolic total extract and ethyl acetate fraction of Xanthocerais lignum exhibited a higher similarity in terms of their TIC profiles.Furthermore, these fractions demonstrated a significantly higher number of chromatographic peaks and superior peak shapes compared to the n-butanol fraction and water fraction.UPLC-Q-Exactive Orbitrap MS analysis identified a total of 71 compounds from the various extracts of Xanthocerais lignum, including 48 in the ethanol total extract, 46 in the ethyl acetate fraction, 45 in the n-butanol fraction, and 35 in the water fraction.Detailed information on the identification of chemical constituents is shown in Table 1.The characteristic components of Xanthocerais lignum are flavan-3-ol and flavanone compounds, in addition to proanthocyanidins formed by the polymerization of flavan-3-ol.The mass spectral fragmentation patterns of compounds such as epicatechin, dihydromyricetin, and proanthocyanidin A2, which have the highest relative content in their respective compound classes, are used to illustrate the mass spectral fragmentation patterns of that class of compounds, with specific fragmentation pathways shown in Figures 3-5

Optimal Blood Sampling Time
The chromatograms of each blood sampling time point are shown in Figure 6A, while the total peak areas at each time point are presented in Figure 6B.By comparing the chromatograms at different time points, it was observed that at 3 h after administration, the chromatographic peaks exhibited excellent peak shape and separation, and the highest number of chemical components were absorbed into the bloodstream (highest number of chromatographic peaks).Additionally, this time point represented the first peak of drug absorption after administration (the first peak in Figure 6B).To accurately identify more components of Xanthocerais lignum in the bloodstream, the time point of 3 h after administration was ultimately selected as the optimal blood sampling time.

Constituents Absorbed into Blood from Xanthocerais lignum
Figure 7A-D illustrates the comparison of TIC between blank control rat serum, serum from rats treated with various Xanthocerais lignum extracts (ethanol, ethyl acetate, n-butanol, and water), and different classes of Xanthocerais lignum extracts.It can be observed that the TIC of the rat serum after administration shows a significant increase in the number of chromatographic peaks compared to the blank control rat.Among them, the ethyl acetate fraction exhibits the highest number of chromatographic peaks.A total of 36 constituents absorbed into blood were identified, with 24, 27, 14, and 8 components detected in the serum of rats treated with the ethanol total extract, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively.Among these, 17 components corresponded to prototype compounds directly entering the bloodstream, while the remaining 19 components were metabolites.Detailed information on the identification of constituents absorbed into the blood from Xanthocerais lignum is provided in Table 2, and peak areas are listed in Table S1.The results indicate that the metabolic pathways of Xanthocerais lignum's chemical constituents predominantly involve glucuronidation.Characteristic metabolites identified include flavonoid glucuronides such as epicatechin-3 ′ -O-glucuronide and 4 ′ -O-methyl-epicatechin-3 ′ -O-glucuronide.By correlating the referenced literature [10,11] with mass spectrometry data, the metabolic trajectory of epicatechin within the body is postulated, as depicted in Figure 7E.Epicatechin undergoes glucuronidation to form epicatechin-3 ′ -O-glucuronide, followed by methylation to further generate 4 ′ -O-methyl-epicatechin-3 ′ -O-glucuronide.

Results of Serum Spectrum-Effect Correlation Analysis
The results of the serum spectrum-efficacy correlation analysis are shown in Table 3.The gray correlation values range between 0 and 1, with higher values indicating stronger correlations with the parent sequence (Y) [12], i.e., the stronger the relationship between the compound and the anti-RA efficacy.Among the top 10 compounds, 7 are prototype blood-entering components of Xanthocerais lignum, while the remaining 3 are metabolites resulting from glucuronidation of flavonoids found in Xanthocerais lignum.These compounds exhibit gray correlation values above 0.8, suggesting their potential for effective anti-RA activity.Following the "principles for determining Q-Markers of Chinese medicine" [8], 4 components were selected as Xanthocerais lignum's anti-RA Q-markers: proanthocyanidin A2, epicatechin, dihydroquercetin, and catechin.These compounds are characteristic chemical constituents of Xanthocerais lignum and exhibit relatively high content.The serum spectrum-effect correlation analysis results also suggest that these 4 compounds may possess potential anti-RA activity.All 4 compounds were further identified by com-parison with the standards, and the chromatogram of the Xanthocerais lignum total extract at 230 nm is shown in Figure 8A.From the ChEMBL database, we have collated 11 experimentally active targets for Q-Marker, with detailed information presented in Table S2.Utilizing the SuperPred database, we have predicted 70 potential targets for Q-Marker.By amalgamating the experimentally active targets with the predicted targets and eliminating duplicates, we have identified 33 unique targets.An aggregate of 6592 RA-related targets was gathered from three databases, among which 21 intersect with the Q-Marker's targets, as depicted in Figure 8B.KEGG enrichment analysis yielded 33 signaling pathways, and the top 10, based on Count values, were selected for visual analysis, the results of which are illustrated in Figure 8C.These signaling pathways are closely associated with the developmental processes of RA.For instance, the HIF-1 signaling pathway can facilitate the formation of osteoclasts, thereby exacerbating inflammatory bone resorption [13].Aberrant activation of the Ras signaling pathway can lead to the destruction of RA synovial tissue and inflammation [14].Activation of the MAPK signaling pathway can induce abnormal proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) [15].Integrating the enrichment analysis parameters with the RA pathogenesis mechanism, we ultimately selected the HIF-1 signaling pathway for subsequent validation.Within the HIF-1 signaling pathway, the Q-Marker of Xanthocerais lignum against RA may exert pharmacological effects such as inhibiting angiogenesis by targeting ERK, NF-κB, HIF-1α, and VEGF, thereby mitigating the progression of RA, with the detailed mechanism of action delineated in Figure 8D.

Molecular Docking Outcomes
Table 4 delineates the molecular docking results of Xanthocerais lignum's anti-RA Q-Markers with 4 targets within the HIF-1 signaling pathway.Proanthocyanidin A2 demonstrated the lowest binding energy with ERK and VEGF targets, epicatechin with NF-κB, and catechin with HIF-1α.The ligand-protein interactions of these compounds are illustrated in Figure 9. Proanthocyanidin A2 formed hydrogen bonds at positions 31 (isoleucine), 32 (glycine), and 108 (methionine) within the A-chain of the ERK protein; it also formed 2 hydrogen bonds at positions 75 (asparagine) and 95 (serine), and 1 hydrogen bond at positions 38, 78 (glutamic acid), and 96 (phenylalanine) within the W-chain of the VEGF protein.Epicatechin established 2 hydrogen bonds at positions 68 (glycine) and 1 hydrogen bond at positions 58 (phenylalanine), 65 (proline), 67 (histidine), 115 (valine), and 142 (isoleucine) within the P-chain of the NF-κB protein.Catechin interacted through hydrogen bonds with phenylalanine at position 15 and aspartic acid at position 17 within the B-chain of the HIF-1α protein.In addition to hydrogen bonds, the complexes exhibited π-cation interactions, van der Waals forces, and other non-covalent interactions, which are crucial in determining the stability and binding affinity of the ligand-protein complexes.From the docking results, it can be observed that the ligand-protein complex with the lowest binding affinity forms a minimum of 2 hydrogen bonds.Compared to other non-covalent interactions, hydrogen bonds make a greater contribution to the stability of the complex.Generally, binding affinity is determined by the free energy of binding, with a stable binding conformation correlating with lower binding energy.A binding energy less than 0 indicates that the ligand can spontaneously bind to the receptor, with smaller values signifying higher binding activity and ease of drug-receptor interaction [16].The negative binding energies across all ligand-protein interactions suggest a favorable binding affinity, further corroborating the mechanism through which Xanthocerais lignum's Q-Markers exert anti-RA effects.The active components present in Xanthocerais lignum may potentially inhibit the activity of the aforementioned proteins by forming complexes with them, thereby exerting an anti-RA effect.

Discussion
The CIA (collagen-induced arthritis) model was initially established in Wistar and Wistar-Lewis rats using type II collagen immunization and was later expanded to include mice and non-human primates [17].Due to their similar immune and pathological characteristics to RA, these CIA models have been widely employed in human RA research.Typically, the construction of the CIA model in rats requires animals that are at least 7-8 weeks old when their immune systems have matured.Wistar rats are a wellestablished strain highly susceptible to CIA, and in our study, approximately 70% of the rats successfully developed the CIA model, making them suitable for subsequent screening of Q-Markers from Xanthocerais lignum with potential anti-RA properties.
Differences were observed in the identified compounds among the various extracts of Xanthocerais lignum.The relative content of flavonoid aglycone was significantly higher in the ethanolic total extract and ethyl acetate fraction compared to the n-butanol fraction and water fraction.This disparity may be attributed to the higher extraction efficiency of ethyl acetate during the extraction process, effectively enriching the flavonoids in Xanthocerais lignum.Numerous studies have demonstrated the favorable anti-RA activity of flavonoids such as quercetin and epigallocatechin gallate.The mechanisms underlying their anti-RA effects primarily involve anti-inflammatory, antioxidant, and immunomodulatory actions [18].The significant anti-RA activity observed in the ethyl acetate fraction is closely associated with its rich content of flavonoids.Flavonoid compounds exhibit greater selectivity and sensitivity in negative ion mode during LC-MS analysis [19], resulting in superior chromatographic separation and signal intensity in the TIC chart when using negative ion mode.
The total ethanol extract of Xanthocerais lignum showed an in vivo absorption trend that initially increased, then decreased, and subsequently increased again over time.This trend may be attributed to the complex chemical composition of the traditional Chinese medicine extract, which could involve multiple drug interactions affecting absorption, distribution, metabolism, and excretion within the body.Moreover, different chemical components may exhibit distinct pharmacokinetic characteristics [20].For instance, some components may be rapidly absorbed but also quickly metabolized and excreted, leading to an initial increase followed by a decrease in their exposure levels within the body.Conversely, other components may be absorbed more slowly, yet their metabolism and excretion are also slow, resulting in a later increase in exposure levels.Due to the complexity of herbal ingredients, studying their pharmacokinetics poses significant challenges.Currently, a widely accepted approach is to represent the overall pharmacokinetic behavior of herbal medicine by investigating the pharmacokinetic characteristics of one or several known active constituents [21].For example, the pharmacokinetics of 2α-hydroxyl-3βangeloylcinnamolide can be studied to represent the pharmacokinetic profile of Polygonum jucundum, while the pharmacokinetic characteristics of 2,3,5,4 ′ -tetrahydroxystilbene-2-Oβ-D-glucoside can be used to associate with Polygonum multiflorum [22,23].Our research indicates that the compounds proanthocyanidin A2, epicatechin, dihydroquercetin, and catechin exhibit potential anti-RA activity.Furthermore, all 4 compounds can be quantitatively analyzed in both the blood of rats and the extracts of Xanthocerais lignum using UPLC.Thus, the pharmacokinetic characteristics of Xanthocerais lignum can be characterized by conducting pharmacokinetic studies on one or more of these compounds.
Multiple constituents of Xanthocerais lignum can be absorbed by rats and enter the bloodstream, with the prototype blood-entering components being organic acids and flavonoid compounds.Some of these are metabolic products within the organism or substances involved in metabolism, such as uric acid, an end product of purine metabolism that serves as an antioxidant protecting cells from free radical damage [24].Citric acid is a crucial intermediate in the tricarboxylic acid cycle, participating in energy metabolism and biosynthesis [25].Methylmalonic acid, an intermediate product of branched-chain amino acid metabolism, can be converted into succinyl-CoA by methylmalonyl-CoA mutase, entering the tricarboxylic acid cycle [26].These compounds are involved in metabolic processes that are widespread in both animals and plants; hence, they can be detected in both Xanthocerais lignum and rat serum.The remaining prototype blood-entering components are secondary metabolites of plants, primarily flavan-3-ol and flavanone compounds, which are relatively abundant in Xanthocerais lignum and possess various pharmacological activities such as antioxidant, anti-inflammatory, and antitumor effects [27].
Some of the flavonoid components in Xanthocerais lignum are converted to flavonoid glucuronides in vivo.Glucuronidation is an important phase II metabolic reaction catalyzed by glucuronosyltransferases, responsible for the clearance of various endogenous and exogenous substances within the body, significantly influencing the efficacy and adverse reactions of drugs [28].Most drugs become more water-soluble and have less pharmacological activity after glucuronidation.Studies have shown that the glucuronidated products of (−)-epicatechin exhibit reduced antioxidant activity compared to the original components [29].
In the serum spectrum-effect correlation analysis, the top 10 ranked compounds are predominantly plant flavonoids with antioxidant, anti-inflammatory, and immunomodulatory effects.They can inhibit the production of RA-related cytokines and inflammatory mediators, such as TNF-α and IL-6, and reduce joint inflammation and damage by suppressing synovial cell proliferation and migration, as well as osteoclast differentiation [30].Additionally, in vitro and in vivo experiments have confirmed the efficacy of these compounds against RA.For example, procyanidin A2 can target the NF-κB, MAPK, and Nrf2 pathways to exert anti-inflammatory and antioxidant effects [31].Catechins can reduce secondary inflammation in rats by inhibiting the production of IL-1, TNF-α, and PGE2 in an adjuvant-induced arthritis model and by upregulating the expression of EP2 in rat synovial cells [32].Epicatechin significantly inhibits the activation of the NLRP3 inflammasome and the NF-κB signaling pathway by inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α) both in vitro and in vivo, thereby exerting anti-inflammatory effects [33].Dihydroquercetin can inhibit RANKL-induced osteoclast differentiation and gene expression, including TRAP and MMP-9, showing potential for treating osteoporosis, bone resorption, and related diseases such as RA [34].
Based on the core theory of "Chinese medicine efficacy-pharmacodynamic material basis-quality control characteristic components", 4 Q-Markers of Xanthocerais lignum have been identified.These markers satisfy the unique specificity, measurability, and effectiveness principles of Q-Markers and can serve as quality indicators for Xanthocerais lignum's anti-RA properties.The relative contents of the 4 Q-Markers in Xanthocerais lignum can be used to preliminarily evaluate the strength of its anti-RA activity.HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits, primarily responsive to changes in oxygen concentration.In RA, the hypoxic microenvironment of the joints is the main inducer of HIF-1 [35].HIF-1 can exacerbate the RA process by promoting inflammation, angiogenesis, and cartilage destruction [36], with the regulation of VEGF gene expression by HIF-1α being a key factor in promoting angiogenesis [37].HIF-1α is regulated by various signaling pathways, and inhibiting the activation of the MAPK and NF-κB signaling pathways can reduce the expression of HIF-1α, thereby alleviating the RA process [36,38].Existing research suggests that elevated expression of HIF-1α facilitates the migration and invasion of RA-FLS, thereby exacerbating the erosion of surrounding cartilage [39].It is postulated that the 4 Q-Markers found in Xanthocerais lignum may exert anti-RA effects by inhibiting the activation of the HIF-1 signaling pathway.

Experimental Animals
Female Wistar rats, aged 7-8 weeks, weighing 200-220 g, totaling 20 individuals, were procured from SiPeiFu (Beijing) Biotechnology Co., Ltd.(Beijing, China), with license number SCXK (Beijing) 2019-0010.The experimental animals were housed in an SPF environment with unrestricted access to water and food.All animal experiments in this study were approved by the Medical Ethics Committee of Inner Mongolia Medical University (approval number: YKD202301197) and adhered to internationally accepted principles for laboratory animal use and care.

Preparation of Various Extracts of Xanthocerais lignum 4.4.1. Preparation of Total Ethanol Extract
The dried Xanthocerais lignum medicinal material, weighing 1 kg, is pulverized and passed through a number 4 sieve (Sieve inner diameter: 250 µm ± 9.9 µm).Subsequently, the powdered material is added to an 8 L solution of 70% ethanol.Using an ultrasonic extraction and concentration device, the material is extracted 3 times at a temperature of 80 • C, with a power of 250 W and a frequency of 40 kHz, with each extraction lasting 2 h.The extracted liquids are combined, and the alcoholic content is removed through vacuum concentration using a rotary evaporator until no alcohol odor remains.The resulting extract is then subjected to freeze-drying, yielding a powdered form of the ethanol extract of Xanthocerais lignum.

Preparation of Different Polarity Extracts
The aforementioned total ethanol extract of Xanthocerais lignum (Z) was suspended in water and then successively extracted with 1.5 volumes of ethyl acetate and n-butanol solutions, 3 times each.The respective extracts were combined, and the different polarity extracts and water fractions were concentrated under reduced pressure and then freezedried to obtain freeze-dried powders of the ethyl acetate fraction (YY), n-butanol fraction (ZDC), and water fraction (S) of Xanthocerais lignum.

Study of Chemical Constituents of Xanthocerais lignum 4.5.1. Preparation of Test Sample Solutions
Precisely weigh the freeze-dried powders of the total ethanol extract, ethyl acetate fraction, n-butanol fraction, and water fraction of Xanthocerais lignum to 0.1001 g, 0.1035 g, 0.1005 g, and 0.1004 g, respectively.Dissolve each powder in chromatographic methanol and make up 10 mL in a volumetric flask.Centrifuge each solution at 16,000 r•min −1 for 10 min at 4 • C and filter the supernatant through a 0.22 µm microporous membrane to obtain the test sample solution.

Identification of Chemical Constituents
Qualitative analysis of the test sample solutions was performed using UPLC-Q-Exactive Orbitrap MS technology.Initially, a compound library of Xanthocerais lignum was established based on existing literature reports (relative molecular mass and secondary mass spectral information were obtained from PubChem and MassBank, respectively).The primary and secondary mass spectral information for each chromatographic peak was searched and matched by combining the in-house library in Compound Discoverer 3.1 (includes compound name, relative molecular mass, and secondary mass spectral informa-tion) with the Xanthocerais lignum compound library.For chromatographic peaks without matching data, the molecular formula of each peak was preliminarily determined based on the actual measured relative molecular mass and the theoretical exact relative molecular mass.The compounds were searched in MassBank (www.massbank.jp,accessed on 5 April 2024) and mzCloud (www.mzcloud.org,accessed on 5 April 2024) databases according to their molecular formula or relative molecular mass.The primary and secondary mass spectral fragmentation data of the chromatographic peak were compared with the compounds in the databases, and the chemical structures were identified in conjunction with the mass spectral fragmentation patterns of this class of compounds.

Animal Modeling and Grouping
After 1 week of acclimatization, a CIA rat model was constructed.The modeling procedure involved dissolving bovine type II collagen in 0.05 mol•L −1 glacial acetic acid, resulting in a final concentration of 2 mg•mL −1 .The dissolved collagen was then refrigerated overnight at 4 • C. The following day, it was mixed with an equidose of Freund's incomplete adjuvant and thoroughly emulsified at 4 • C. The CIA rat model was established by subcutaneously injecting a total volume of 0.2 mL of the collagen emulsion into the base of the tail and the footpad.Immune enhancement was achieved after 1 week by injecting an additional 0.1 mL of the collagen emulsion using the same method.After 3 weeks of initial immunization, 14 out of the 20 rats exhibited complete redness and swelling in all of their paws, including the ankle joints, signifying successful modeling.From this group of rats, 12 rats were randomly selected and assigned to different treatment groups: the total ethanol extract of Xanthocerais lignum (Z), ethyl acetate fraction (YY), n-butanol fraction (ZDC), and water fraction (S).Each group consisted of 3 rats, while the remaining 2 rats were used as blank controls and for optimizing the blood collection time, respectively.All rats were fasted for 12 h but allowed access to water before oral administration of the drug.The freeze-dried powders of the ethanol extract of Xanthocerais lignum, ethyl acetate fraction, n-butanol fraction, and water fraction were suspended in a 0.5% CMC-Na solution.The administration was conducted according to the designated groups, with a dosage of 1.35 g•kg −1 for each group (human clinical dosage was 3 g•d −1 , which was 5 times the human clinical daily dose based on body surface area).Administration continued for 3 consecutive days to enhance the response of the blood-entering components of Xanthocerais lignum.The blank control rats received an equivalent volume of CMC-Na solution for 3 consecutive days.After the final administration, approximately 0.6 mL of blood was collected from each rat via the jugular vein.The collected blood was allowed to rest at room temperature for 1 h and then centrifuged at 5000 rpm for 10 min at 4 • C. A 200 µL aliquot of the supernatant was mixed with 800 µL of methanol and vortexed for 30 s to precipitate proteins.The mixture was further centrifuged at 15,000 rpm for 10 min at 4 • C to separate the proteins.The supernatant was collected and evaporated using a centrifugal concentrator.Subsequently, 200 µL of methanol was added, followed by vortexing for 2 min.The mixture was then centrifuged at 16,000 rpm for 10 min at 4 • C, and the supernatant was filtered through a 0.22 µm microporous membrane to obtain the serum test solution.

Chromatographic and Mass Spectrometric Conditions
As per Section 4.5.2, the sample volume for each group of rat serum test samples was 10 µL.

Optimization of Optimal Blood Collection Time
Blood was collected before administration and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 h after administration of Xanthocerais lignum ethanol total extract.The serum test sample solution was prepared according to the method in Section 4.7.1.The optimal blood sampling time is determined based on the number of chromatographic peaks, peak areas, and separation degree at each time point.

Identification of Prototype Blood-Entering Components and Metabolites
The serum test sample solution was prepared according to the method in Section 4.7.1 (blood collected at the optimal time point), and UPLC-Q-Exactive Orbitrap MS technology was used for detection.Compound identification is performed according to the method outlined in Section 4.5.3.By comparing the blood components of the blank control rats with those of the medicated group rats, combined with the chemical components of Xanthocerais lignum, the prototype blood-entering components and metabolites of Xanthocerais lignum are clarified.

Serum Spectrum-Effect Correlation Analysis of Xanthocerais lignum
Our research group previously evaluated the anti-RA effects of various extracts of Xanthocerais lignum through multiple methods, such as joint swelling measurement, tissue pathology observation, and cytokine level detection, from holistic, organ-tissue, and molecular levels.The therapeutic effect of each administration group on arthritis in rats is shown in Figure 10.These pharmacological data are reverse-normalized (after mapping the data uniformly to the range [0, 1], the values are subtracted from 1 to ensure that the pharmacological information and the therapeutic efficacy of the drugs are positively correlated) and assigned different weights (0.4 × holistic level efficacy + 0.3 × organ-tissue level efficacy + 0.4 × molecular level efficacy) as dependent variables (Y) in the spectrum-effect correlation analysis.The peak areas of the constituents absorbed into the blood from various extracts of Xanthocerais lignum were used as independent variables (X) for grey relational analysis.The anti-RA pharmacological information of various extracts of Xanthocerais lignum is shown in Table S3.During the analysis, mean normalization was used as a dimensionless treatment method (data of the parent sequence and the characteristic sequence were divided by their respective mean value), with a resolution coefficient ρ = 0.5 (the smaller the resolution coefficient is, the larger the resolution is, and the best resolution is achieved at ρ ≤ 0.5463, usually set at ρ = 0.5).The gray correlation values are obtained by solving the gray correlation coefficient between the parent sequence (Y) and the characteristic sequence (X), and the pharmacodynamic material basis of Xanthocerais lignum against RA was determined based on the ranking results of the gray correlation values.

Prediction of the Mechanism of Q-Markers of Xanthocerais lignum against RA
After determining the pharmacodynamic material basis of Xanthocerais lignum against RA, the Q-Markers of Xanthocerais lignum against RA were screened based on the "principles for determining Q-Markers of Chinese medicine" [8], and then compared with standards.The screening criteria are as follows: (1) Effectiveness: compounds ranked in the top 10 based on serum spectrum-effect correlation analysis.(2) Measurability: compounds detectable in both Xanthocerais lignum herbal material and rat serum.(3) Specificity: characteristic chemical components in Xanthocerais lignum, primarily belonging to the flavanols and flavanone classes.
Experimental activity targets of Q-Markers were collected from the ChEMBL database (www.ebi.ac.uk/chembl, accessed on 21 April 2024), and potential action targets of Q-Markers were predicted using the SuperPred database (https://prediction.charite.de,accessed on 21 April 2024), with a selection criterion of Probability > 80%.The Gene ID Conversion Tool in the DAVID database (https://david.ncifcrf.gov,accessed on 21 April 2024) was used to convert "Uniprot ID" to "Official Gene Symbols".
RA-related targets were collected from the Gene Cards (www.genecards.org,accessed on 21 April 2024), DisGeNET (www.disgenet.org/home,accessed on 21 April 2024), and OMIM (www.omim.org,accessed on 21 April 2024) databases.After intersecting the disease targets with the Q-Marker targets, the overlapping targets were imported into the DAVID database for KEGG enrichment analysis.The p-values were corrected using the false discovery rate (FDR) control method (FDR < 0.05).Ultimately, significant signaling pathways were selected based on a threshold of P < 0.05 to predict potential action pathways and targets of Q-Markers in the context of RA treatment with Xanthocerais lignum.

Validation by Molecular
Docking Protein receptor structures were obtained from the PDB database (www.rcsb.org, accessed on 22 April 2024), and small molecule ligand structures were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov,accessed on 22 April 2024).After processing the receptor and ligand molecules using Discovery Studio 2019, molecular docking was performed using the CDOCKER method.For protein receptor complexes with inherent small molecule ligands, binding sites were generated at the location of the ligand molecule; if the protein receptor did not contain a small molecule ligand, binding sites were sought within the receptor cavity.After docking, the CDOCKER interaction energy was calculated, and the compound with lower binding energy was selected for analysis of its ligand-protein interactions.

Conclusions
In this study, 15 flavonoid compounds were identified from Xanthocerais lignum, primarily belonging to the classes of flavanols and dihydroflavones.Among them, 7 compounds were observed to be absorbed into the bloodstream in their original forms, while 4 compounds underwent metabolism via the glucuronidation pathway.Serum spectrum-effect correlation analysis results suggest that procyanidin A2, dihydroquercetin, catechin, epicatechin, and their glucuronidated products may possess potential anti-RA activity, serving as the pharmacodynamic material basis for Xanthocerais lignum's anti-RA effects.Based on these findings, 4 components were selected as Q-Markers for Xanthocerais lignum's anti-RA properties.The action mechanisms of these components, as predicted using compoundtarget interaction data and molecular docking techniques, may involve modulating the expression of proteins such as ERK, NF-κB, HIF-1α, and VEGF within the HIF-1 signaling pathway.This modulation may exert pharmacological effects that inhibit inflammation, angiogenesis, and cartilage destruction, thereby alleviating the progression of RA.However, the detailed mechanisms of action require further validation through in vitro and in vivo experiments.For instance, in vitro assessments can be conducted using human or animal cell lines pertinent to RA to examine the impact of Q-Markers on key proteins within the HIF-1 signaling pathway, such as ERK, NF-κB, HIF-1α, and VEGF.Furthermore, in vivo studies utilizing CIA models can be conducted to observe the therapeutic effects of the Q-Markers, including the evaluation of inflammatory cytokines, angiogenesis, and joint pathological changes.Xanthocerais lignum has broad application prospects for anti-RA treatment.This study provides valuable references for the quality assessment of Xanthocerais lignum and the further development of lead compounds with anti-RA activity derived from Xanthocerais lignum.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/molecules29133191/s1,Table S1: Peak areas of migrating components in the blood of various extracts of Xanthocerais lignum; Table S2: Experimentally active targets of Q-Marker; Table S3: Anti-RA efficacy of various extracts of Xanthocerais lignum.
. Epicatechin loses one molecule of CO 2 , resulting in a fragment ion at m/z 245.It also loses two molecules of C 2 H 2 O, producing a fragment ion at m/z 205.Additionally, it undergoes B-ring cleavage, generating a fragment ion at m/z 179.Furthermore, on the basis of the 179-fragment ion, it loses one molecule of CO, resulting in a fragment ion at m/z 151.Catechin also undergoes retro-Diels-Alder (RDA) reaction, leading to a fragment ion at m/z 137.Moreover, C-ring 1,4 bond breakage produces a fragment ion at m/z 125; Dihydromyricetin primarily undergoes B-ring cleavage and RDA cleavage, producing fragment ions at m/z 193 and m/z 151, respectively; Proanthocyanidin A2, similarly, mainly undergoes B-ring cleavage and RDA cleavage, resulting in fragment ions at m/z 449 and m/z 423, respectively.Additionally, it can lose one molecule of epicatechin, generating a fragment ion at m/z 289.

Figure 7 .
Figure 7. Serum TIC plots of rats with compound metabolic pathways: (A) serum TIC plots of rats in the ethanol extract group of Xanthocerais lignum; (B) ethyl acetate fraction group; (C) n-butanol fraction group; (D) water fraction group; (E) possible in vivo metabolic pathways of epicatechin.

Figure 8 .
Figure 8. Identification of Q-Marker for anti-RA in Xanthocerais lignum and their mechanisms of action: (A) chromatogram of the total extract of Xanthocerais lignum; (B) intersection of Q-Marker targets with RA disease targets; (C) results of KEGG enrichment analysis; (D) possible mechanism of anti-RA action of Q-Marker.

Figure 10 .
Figure 10.Therapeutic effect of each administration group on CIA rats (CK: blank control group; M: CIA model group; JA: methotrexate group).

Author
Contributions: H.Q.: Conceptualization, Methodology, Data curation, Writing-original draft; L.S.: Data curation, Formal analysis; Y.Y.: Investigation, Validation; X.T.: Resources, Formal analysis; Q.D.: Project administration, Writing-review and editing; F.M.: Software, Writing-review and editing; X.W.: Conceptualization, Funding acquisition, Supervision, Writing-review and editing.All authors have read and agreed to the published version of the manuscript.Funding: This work was financially supported by [National Natural Science Foundation of China] (Grant number 82360835) and [Natural Science Foundation of Inner Mongolia] (Grant number 2022MS0826).Institutional Review Board Statement:All animal experiments in this study were approved by the Medical Ethics Committee of Inner Mongolia Medical University (approval number: YKD202301197) and adhered to the internationally accepted principles for laboratory animal use and care.Informed Consent Statement: Not applicable.

Table 1 .
Chemical constituents of each extract of Xanthocerais lignum.

Table 2 .
Migratory components in the blood of various extracts of Xanthocerais lignum.

Table 3 .
Results of gray correlation analysis.